Methods and systems for processing samples on porous substrates

ABSTRACT

Methods and systems for processing samples fixed to a porous substrate generally comprising, a compressor defining one or more fluid isolation areas, a support, for the porous substrate, having an opening corresponding to one or more of the fluid isolation areas of the compressor, an actuator that causes at least a portion of the compressor to press against the porous substrate, a fluid inlet having access to the fluid isolation area at least when the compressor is pressed against the porous substrate, and a fluid outlet to receive fluid, through the opening in the support corresponding to the fluid isolation area of the compressor, at least when the compressor is pressed against the porous substrate.

BACKGROUND

The invention relates generally to methods and systems for processingsamples on porous substrates.

Porous substrates, such as cellulose matrices (e.g. 31 ETF, FTA and FTAelute cards available from Whatman) are often used to store biologicalsamples, such as blood. A new application area for these cards is in thepharmaceutical industry, which is using them to store dried bloodsamples from pharmacokinetic and toxicokinetic studies. When it is timeto analyze the amount of drug or drug metabolite in the dried bloodspot, the current methods require the user to cut the sample out of thecard, usually a 1-6 mm diameter circle, place the cut disc in a vial orwell with extraction fluid, and then shake/vortex for a set period oftime. The extraction fluid is then removed and analyzed using a methodsuch as LC-MS.

The pharmaceutical industry is expecting to process a large number ofsamples per day and is therefore looking for ways to automate theprocess. The current workflow of disc cutting and extraction, posesseveral problems when facing the challenge of automation. The primaryproblems arise from the cutting step. The small cut discs are highlyprone to the effects of static electricity or even a light breeze. Thereare numerous reports of cut discs being lost during the cutting step orduring transport of the cut discs. Cross-contamination is anothersignificant problem associated with having to cut pieces out of the FTAcards because small fibers are often released during cutting. Thesesmall fibers can then cause cross-contamination between samples.

Previous attempts to automate the workflow include cutting out a portionof the card with sample dried on it. The cut disc is then placed in avial/well, to which extraction fluid is added, and then shaken/vortexedfor a set period of time. Alternatively, the cut disc is placed in adevice that allows one to flow fluid through it to extract analytes. Allof these approaches suffer from the problems and risks associated withcutting (e.g. lost sample discs, loose fiber contamination,contamination from the cutting blade).

Another approach has been to pre-cut a portion of a blank card, placethe sample on the pre-cut disc of substrate, and then extract from theentire disc (by vortexing, shaking, or flow-through). Although thisprocess addresses some of the risks associated with cutting (e.g. thecutting is done before sample application), it has limited applicationof use and does not allow one to analyze the sample multiple times. Theuses of this method are limited because of the dependence of thisprocess on the amount of blood fixed to the card. If the amount ofsample applied to the precut disc is not consistent, the amount of drugor drug metabolite will also not be consistent. There are manysituations and environments where it is difficult to achieve an accurateand consistent amount of sample collection. Inconsistency may, forexample, be due to the manner in which the sample is collected (i.e.fingerstick for blood) or the training level of the people collectingthe samples.

Another approach has been to place the card on a hard surface and thenpress down with a circular knife-edge, which presses against card butdoes not cut through it. Extraction buffers are then passed over thesurface of the card that is isolated by the knife-edge. This methodavoids cutting, but does not ensure that the fluid extracts from thefull depth of the card (e.g. only the analytes at the surface may beextracted). It also does not provide a way to remove the fluid from theisolated area of the card before removing the knife-edge. This couldlead to fluid wicking into the surrounding area after the knife-edge isremoved. This would damage the remaining sample making re-sampling fromanother position on the card difficult or impossible. Also, when flowingfluid over the top of the sample, fibers are sometimes released. Thisapproach also uses an in-line frit to remove released fibers. However,these fits may become clogged overtime and cause cross-contamination andthus must be cleaned or replaced between samples and this requiresadditional steps from the user.

BRIEF DESCRIPTION

The methods and systems of the invention generally relate to extractinganalytes from samples dried onto porous substrates (such as a cellulosecard) for analysis of drug and drug metabolites in body fluids. Themethods generally use compression to isolate an area of the card throughwhich an extraction buffer is then flowed through, perpendicular to thesurface of the card. The method may comprise a clearing step to removeremaining extraction buffer from the card and system. This methodovercomes the risks and problems associated with cutting the paper priorto extraction. By flowing the fluid perpendicular to the paper withinthe compression seal, this ensures that the extraction is uniform acrossthe isolated area. Performing additional extractions from differentpositions on the blood spot is also possible with these methods. Forexample, the clearing step ensures the integrity of the sample aroundthe isolated area is maintained when the compression force is removed.

The methods and system also allow easy integration of a disposablemembrane to remove any fibers that are released as fluid flows throughthe cellulose substrate. The membrane may be added and replaced withoutany additional steps by the user, facilitating the connection of thesystem directly to an analysis system such as a mass spectrometer.

The methods and systems of the invention overcome several of thechallenges in automating the use of cellulose matrix cards such as FTAand FTA Elute. The methods and systems allow one to extract from adefined area of the porous substrate without having to cut. Thiseliminates the risk of losing a cut disc or creating loose fibers duringcutting that could contaminate another sample. It also enables more thanone test to be applied to, not only the entire sample on the card, butalso to that portion of the sample isolated by compression. The methodsand systems result in a consistent quantity of sample being tested,because they effectively isolate a defined area of the sample. Forexample, even if the amount of blood collected on the paper varies, themethods are always extracting/analyzing the same area of dried blood.For products, such as the Whatman cards, where the sample wicks outuniformly, analyzing the same area is equivalent to analyzing the samevolume of the original sample material. Improving the automationsolutions available for cellulose card handling supports the growth ofthese materials in fields such as the pharmaceutical industry which thatrequire high-throughput analysis.

An example embodiment of the system of the invention, for processingsamples fixed to a porous substrate, comprises: a compressor definingone or more fluid isolation areas; a support, for the porous substrate,having an opening corresponding to the fluid isolation area of thecompressor; an actuator that causes at least a portion of the compressorto press against the porous substrate; a fluid inlet having access tothe fluid isolation area at least when the compressor is pressed againstthe porous substrate; a fluid outlet to receive fluid, through theopening in the support corresponding to the fluid isolation area of thecompressor, at least when the compressor is pressed against the poroussubstrate. The system may further comprise a compressible membrane orfilter, to remove fibers. The membrane or filter is placed between theporous substrate and the compressor so that the fluid exiting the poroussubstrate travels through the membrane or filter before entering thefluid outlet.

The compressor may comprise a top seal plug and a bottom seal plug, oneor both of which the actuator causes to press against the poroussubstrate; wherein the bottom seal plug has an opening in fluidcommunication with the opening in the support and an opening in fluidcommunication with the fluid outlet. The top seal plug may have anopening in fluid communication with the fluid inlet. The system mayfurther comprise a receptacle comprising a plate having a plurality ofwells and/or vials. The system may also be directly connected to ananalysis system such as a mass spectrometer.

Another embodiment of the system, for processing samples fixed to aporous substrate, comprises: a plurality of sample processingsubsystems, each of which comprises, a compressor defining one or morefluid isolation areas, a support, for the porous substrate, having anopening corresponding to the fluid isolation area of the compressor, anactuator that causes at least a portion of the compressor to pressagainst the porous substrate, a fluid inlet having access to the fluidisolation area at least when the compressor is pressed against theporous substrate, a fluid outlet to receive fluid, through the openingin the support corresponding to the fluid isolation area of thecompressor, at least when the compressor is pressed against the poroussubstrate; a manifold in fluid communication with the fluid inlet of thesample processing subsystems; a cassette for housing a plurality ofporous substrates; a receptacle shuttle subsystem for transporting oneor more fluid receptacles to the sample processing subsystems; anautomated assembly for transporting the porous substrates from thecassette to the sample processing subsystems; and a controller thatcoordinates the sample processing subsystems and the automated assembly.The system may further comprise an imaging subsystem for imaging thesubstrates to identify the location of the sample on the poroussubstrates. The automated assembly may comprise a robotic subassemblyand a guide along which the robotic subassembly moves between thecassette and the sample processing systems. The automated assembly alsotransports the porous substrates to the imaging subsystem.

Another embodiment of the system, for processing samples fixed to aporous substrate, comprises: a compressor defining one or more fluidisolation areas; a support, for the porous substrate, having an openingcorresponding to the fluid isolation area of the compressor; an actuatorthat causes at least a portion of the compressor to press against theporous substrate; a fluid inlet having access to the fluid isolationarea at least when the compressor is pressed against the poroussubstrate; a fluid outlet to receive fluid, through the opening in thesupport corresponding to the fluid isolation area of the compressor, atleast when the compressor is pressed against the porous substrate; and aclearing component that clears one or more of the opening in thesupport, the fluid inlet or the fluid outlet. The clearing component mayclear, for example, by forcing a gas through one or more of the openingsin the support, the fluid inlet or the fluid outlet.

An example of the method for processing samples fixed to a poroussubstrate on a support may comprise: creating a compression seal, on theporous substrate, to form an isolation zone within which a portion ofthe sample is thereby isolated; applying a fluid to the sample isolatedin the isolation zone by flowing the fluid through the isolated zone;collecting at least a portion of the fluid after it is flowed throughthe isolation zone; clearing the fluid from the isolation zone byflowing gas through the isolated zone; releasing the compression seal;and analyzing one or both of the collected fluid and the portion of thesample in the isolation zone. The method may further comprise imagingthe sample on the porous substrate before creating the compression seal,and/or imaging the sample after the fluid is collected. The step ofcreating the compression seal may also comprise compressing acompressible membrane of filter along with the porous substrate. Theanalyzing step may comprise quantifying an amount of one or moresubstances in the collected fluid. In methods in which the samplecomprises blood or other various types of biological materials, theanalyzing step may comprise identifying one or more components of thesample. The step of creating the compression seal may comprise forming aplurality of isolation zones on the porous substrate. A plurality offluids may be applied to the sample simultaneously or serially.

DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood when the following detaileddescription is read with reference to the accompanying drawings in whichlike characters represent like parts throughout the drawings, wherein:

FIG. 1 is a perspective drawing of an embodiment of a compressor of theinvention for processing samples on a porous substrate;

FIG. 2 is a cross-sectional drawing of the embodiment of the compressorshown in FIG. 1;

FIG. 3 is a schematic drawing of an example of the method of theinvention using the compressor shown in FIG. 1;

FIG. 4A is an image showing wicking of fluid on a porous substratewithout the use of a compressor of the invention;

FIG. 4B is an image showing the isolation of fluid within an isolationarea using a compressor of the invention;

FIGS. 5A and 5B are images of the top and bottom, respectively, of asample on a porous substrate showing an area on the sample that has beenisolated using a compressor of the invention;

FIG. 6 is a plot showing the amount of Hyamine extracted from driedblood spots spiked with varying amounts of Hyamine.

FIG. 7 is a perspective drawing of an embodiment of a compressor unitand a fluid unit of the invention for processing samples on a poroussubstrate; and

FIG. 8 is a perspective drawing of an embodiment of a high throughputsystem of the invention for processing samples on a porous substrate.

DETAILED DESCRIPTION

The methods, devices and systems of the invention generally compress anarea of a porous substrate, such as a cellulose card, to isolate aportion of the substrate on which a biological sample has previouslybeen placed, and then pass an extraction buffer through the isolatedportion of the substrate, perpendicular to the plane of the substrate,to extract at least a portion of the biological sample from the card.

Another embodiment of the system for processing samples fixed to aporous substrate comprises: a compressor defining one or more fluidisolation areas; a support, for the porous substrate, having an openingcorresponding to the fluid isolation area of the compressor; an actuatorthat causes at least a portion of the compressor to press against theporous substrate; a fluid inlet having access to the fluid isolationarea at least when the compressor is pressed against the poroussubstrate; a fluid outlet to receive fluid, through the opening in thesupport corresponding to the fluid isolation area of the compressor, atleast when the compressor is pressed against the porous substrate; and aclearing component that clears one or more of the opening in thesupport, the fluid inlet or the fluid outlet. The clearing component mayclear, for example, by forcing a gas or a liquid through one or more ofthe opening in the support, the fluid inlet or the fluid outlet.

Following are non-limiting examples used to illustrate various examplesand embodiments of the methods and systems for processing samples onporous substrates.

Example

As shown in FIGS. 1-3, an embodiment of a compression device, generallyreferred to as device 10, of the invention comprises, a compressor whichmay comprise, for example, the top seal plug 14 and the bottom seal plug18, wherein the top and bottom plugs have a fluid inlet and fluidoutlet, respectively, or channels 15 and 19, with openings that definean isolation area, that allow for the passage of fluid. The channels ofthe plugs of this example are matched in size and aligned so that, ifthe top and bottom plug were in contact with each other, fluid wouldflow through the two parts without obstruction. In this non-limitingembodiment, the flow through the channels has a circular cross sectionwith a 3 mm diameter at the parting line, isolation area, of the twoparts. The channel may narrow, as shown, for example in FIG. 2, in crosssection above and below the parting line of the two parts. The hollowplugs at the parting line, in this non-limiting example, have a sealingarea 42 of 0.0139 in² (9 mm²) to form an isolation area 46 with a 3 mmdiameter cross section. A flangeless tubing nuts 22 (ferrule not shown)is used in this example to support tubing, having an outside diameter of1/16″ and an inside diameter of 1 mm, to transport fluid, such as abuffer through the fluid inlet 26 and outlet 28. The isolation area 46is not limited to 3 mm (arrow C) or to a circular cross section. Thesize and shape of the isolation area may be varied depending on a givenapplication or use of the device.

The top seal plug may be a separate and optionally disposable componentthat is attached directly to the compression actuator or to an actuatorsubassembly. The bottom seal plug may be a separate and possiblydisposable component that is accurately positioned by the base supportstructure. Alternatively, the bottom seal plug could be an integral partof the base support structure.

To use the device, a porous substrate 24, e.g. a cellulose substrate, isinserted between the top seal plug 14 and bottom seal plug 18 into slot16. The top seal plug is associated with an actuator, such as top pusher12, that moves vertically, in this example, or generally perpendicularto the surface of the cellulose substrate. The actuator applies asealing force (shown by arrows A) to the top seal plug relative to thebottom seal plug. The force creates a defined pressure over the areadefined by the areas of the two parts that are in common at the partingline, referred to herein as the sealing area 42 that defines isolationarea 46. The components of the device are seated in a support base 20.When significant pressure is applied to the sealing area, the fibers ofthe cellulose substrate compress and significantly limit the flowparallel on the substrate (wicking) to within the isolation area 46,thus effectively directing any fluid flow through, and generallyperpendicular to, the paper (as shown by arrow B). The bottom seal plug18 has an opening and extends away from the opening to support theporous substrate beyond the area defined by the opening.

The top and bottom seal plugs effectively press against the poroussubstrate (e.g. a cellulose card) to create a barrier of compressedfibers that prevent wicking. Without the barrier of compressed fibers,the majority of the fluid would wick outward and along the surface ofthe paper, rather than flowing perpendicular to it. The isolation area,formed by the compression, allows the system to sample from a definedarea without the need to cut and capture pieces of the substrate onwhich the sample is fixed. For example, there is no need for a step tocut pieces (e.g. discs) of the porous substrate, on which a portion ofthe sample is fixed, and then a step to capture the cut piece in areceptacle, such as a well. By isolating the fluid path, the methods andsystems of the invention eliminate the problems associated with losingthe cut pieces and cross-contamination caused by fibers that come looseduring the cutting and capturing of the cut pieces. To further avoidcontamination between samples, the methods and systems may comprise awash step between analyte extractions, or may use a disposable coverthat is changed between samples (e.g. similar to automated methods andsystems that use disposable pipette tips).

FIGS. 4A and 4B illustrate the difference between applying a buffer to aporous substrate without compression and with compression, respectively.Specifically, in the example shown in 4A, 50 uL of water (with addedfood coloring) was applied to and allowed to flow through 31 Etchromatography paper 30 without any compression applied to the pusherhead. As shown, the fluid only wicked outward along paper, as shown byspot 32, and does not flow through paper. In contrast, in the exampleshown in 4B, 50 uL of water (with added food coloring) was applied toand allowed to flow through 31 Et chromatography paper 34 with 200 lbfof compression is applied to the pusher head. As shown, the fluid didnot wick outside of the isolation area 36. Instead the fluid flowedthrough, and perpendicular to, the paper and within the boundary of theisolation area.

FIGS. 5A and 5B illustrate the effect of using the compression toisolate a sample, showing a top view and a bottom view, respectively, ofa porous substrate 48 (e.g. FTA card) after an example of the method isapplied to a dried blood sample on the substrate. In this example, waterwas applied and allowed to flow through a 3 mm isolation area 49,generally determined by the shape and size of the compression area 47,of a dried blood spot. As shown, the water removed the hemoglobin fromthe spot, as shown by the white, round area of isolation area 49. Thearea of extraction is uniform when view from the top (FIG. 5A) or thebottom (FIG. 5B) of the porous substrate.

Example

An example of one of the methods of the invention for processing sampleson a porous substrate comprises, placing a porous substrate (containingdried analyte) in the slot of the device so that heads of the seal plugsare aligned with the desired extraction area, then applying a force toseal plugs (via pusher). The heads of the seal plugs compress the paperforming a seal which prevents liquids, such as an extraction buffer,that are introduced to the isolation area, via a fluid inlet, fromwicking outward from the initial point at which the buffer is applied tothe substrate. The buffer is applied to the substrate through an inlettube that located, in this example, within a hollow bore concentricallylocated in the first pusher. The buffer that flows through the paper,without wicking outside the isolation area, and then through an outlettube that is located, in this example, within a hollow boreconcentrically located in the bottom, or second, pusher, and into areceptacle such as a well plate or a vial. One or both of the pushers(seal plugs) may move towards the other, or one may be stationary andwhile only the other pusher move towards the stationary pusher. Thefluid in this example flows in a direction that is perpendicular to thepaper. After one or more extractions, the surfaces of the device thatcome into contact with the sample and/or the extraction fluids may becleaned or otherwise cleared of materials to reduce or prevent crosscontamination. For example, air may be introduced into and forcedthrough, the device or system to remove any remaining liquid or foreignmaterials within the fluid path, while the compression force is beingapplied. Air may also be introduced to remove excess fluid from thesample area to dry the location and prevent wicking of fluids after thecompression force is released. Then the compression force is releasedfrom the seal plugs and the paper is removed from the slot. The clearingstep may also be carried out, or repeated, after the paper is removedand the compression forced reapplied to reconnect the fluid path throughthe various components of the device or system.

As a more specific, but non-limiting, example, blood samples weretreated with varying amounts of Hyamine and then 15 μL of the bloodsample was applied to an FTA card. A portion of the blood spot wasisolated, by applying compression around a portion of the blood sample.Then 300 μL of 70% THF was introduced through the fluid inlet andallowed to flow through the blood spot within the isolation area boundedby the compression area (e.g. 3 mm inner diameter) at 60 μL/min. The 70%THF is collected in a vial after exiting the outlet and is then analyzedusing liquid chromatography-mass spectrometry (LC-MS) and calibrationstandards are used to convert peak intensity reading into concentrationdata FIG. 6 shows a plot of the concentration of Hyamine extracted fromeach of the blood spots. There is a linear correlation between theamount of drug spiked into the blood and the amount extracted.

The methods and systems of the invention may analyze the samples andmaterials extracted from the samples for many different purposes using avariety of analyzing systems such as, but not limited to, immunoassays(e.g. to identify the presence or absence of a component), liquidchromatography with UV detection (e.g. to characterize and quantifycomponents), and liquid chromatography with mass spectrometry (e.g. toidentify and/or quantify components).

As another more specific, but non-limiting, example, Proguanil wasspiked into a blood sample at 50 μg/mL and 15 μL, aliquots of the bloodwere applied to an FTA card. A compressible membrane (Pall Life SciencesSupor-200 membrane filter, 0.2 um pore) was placed between an FTA cardwith the dried sample 24 and the bottom seal plug 18. Fluid leaving theisolated area must pass through the membrane before going through theoutlet. The pore size of the membrane is smaller than any fibers thatmay be released. The card and membrane are both compressed in thedevice. Extractions were performed using the compression system with 100μL, of 70% THF. The 70% THF is collected in a vial after exiting theoutlet and is then analyzed using liquid chromatography-massspectrometry (LC-MS) and calibration standards are used to convert peakintensity reading into concentration data. Two replicates wereperformed. Below is a table illustrating the amount of drug detected inthe extraction buffer with and without the membrane. Adding the membranedid not decrease the isolation integrity or extraction efficiency.

With Membrane Without Membrane 0.29 μg/mL, 0.28 μg/mL, 0.31 μg/mL 0.29μg/mL

The methods and systems may be adapted for high-throughput applications.FIG. 7 is an example embodiment of a high-throughput compression device50 for processing samples on porous substrates 70. The device of thisembodiment comprises a compression unit 52, a pusher 54, and a vialstrip 66 on which a plurality of vials 68 are positioned. The systemfurther comprises a fluidic unit 56, for introducing fluidic materialssuch as an extraction buffer, which is in fluid communication with thecompression unit 52 via a fluid path 60. The fluidic device comprises asyringe pump 62, a multi-port valve 64 and a solvent manifold 58. Thedevice may be a stand-alone device or it may be one of several suchdevices in a larger processing system as shown in FIG. 8.

The embodiment of the high-throughput system 80 shown in FIG. 8generally comprises a plurality (e.g. two or more) of compressiondevices 81, comprising for example, the compression unit 52 shown inFIG. 7, in fluidic communication with one or more fluidic devices 56.The solvent manifold 82 may be a single manifold, as shown, thatsupplies solvent from a solvent tank 86 to the fluidic devices viaconnectors 84 in parallel, or may comprise multiple manifolds eachassociated with one or more of the fluidic devices and/or supplyingdiffering materials to the fluidic devices. The system may comprise wellplate shuttles 88 for transporting vial strips along a linear slide orconveyor 90 from a well-plate stacking device 104 to a plurality ofcompression devices 81 in the direction shown by arrow D. Each of thecompression devices may comprise a vial strip shuttle 92 and a verticalaction, linear slide 93 to maneuver vial strips on and off the wellplate shuttle 88, and into a fluidic extraction path of a givencompression device, as shown by arrows E and F. The vials in the vialstrips may comprise pierceable caps for transferring the materialsextracted from the sample spots into the vials. The pierceable caps maybe made of materials that self-repair after piercing to maintain theintegrity of the materials in the vials.

In the embodiment of a high-throughput system shown in FIG. 8, thesystem is an asynchronous parallel processing system that comprises amulti-axis (e.g. three-axes illustrated, for example, by arrows G, H andJ) Cartesian robot 98, with a gripper 100, that maneuvers the FTA cardsfrom a magazine cassette 94, used to support and/or handle multiple FTAcards, to the compression devices. The robot may also be used tomaneuver the FTA cards to one or more imaging position/locationsassociated with an imaging device 96 (e.g. digital camera) so that animage can be taken of the FTA card after (and/or before) one or morematerials are extracted from one or more samples on the FTA cards. Theimages may then be processed to analyze the imaged sample spots on theFTA cards using a detection system (not shown). A controller 102 may beprogrammed to coordinate the various subsystems and devices of system80, including coordination of the plurality of sample processingsubsystems, each of which, in this example embodiment, comprises, acompressor defining one or more fluid isolation areas, a support, forthe porous substrate, having an opening corresponding to the fluidisolation area of the compressor, an actuator that causes at least aportion of the compressor to press against the porous substrate, a fluidinlet having access to the fluid isolation area at least when thecompressor is pressed against the porous substrate, a fluid outlet toreceive fluid, through the opening in the support corresponding to thefluid isolation area of the compressor, at least when the compressor ispressed against the porous substrate. The controller may also coordinatethe timing and introduction of the fluids, such as extraction buffers,via the manifold 82 in fluid communication with the fluid inlets of thesample processing subsystems 81; the movements of automated assembly(e.g. robot 98) for transporting the porous substrates from cassette 94to the sample processing subsystems 81; the movements of the receptacleshuttle subsystem for transporting one or more fluid receptacles (e.g.vials or well plates) transported on shuttle 88 along linear slide 90 tothe sample processing subsystems; and the positioning of each receptacleon strip shuttle 93 into the fluid path of each sample processingsubsystem 81.

Example

A non-limiting example of a process used in connection with system, suchas for example the system shown in FIG. 8, variably may comprise thefollowing actions. A well plate is transferred from a plate stacker. Avial strip is retrieved from the well plate shuttle and a vial on thevial strip is positioned in the fluid path of sample processingsubsystem (e.g. 81). The automated assembly (e.g. 98) retrieves an FTAcard from the magazine cassette and moves the FTA card into the imagingfield of a camera. The imaging system determines the location of one ormore samples on the FTA card. The automated assembly then positions theFTA card within the isolation area and fluid flow path of thecompression device. The actuator of the compression device is extendedto compress the FTA paper. One of the vials is raised to pierce thecover of the vial. Solvent is then pumped from the manifold through thefluid inlet of the compression device and through isolation area of thesample on the FTA card and, together with the extracted materials fromthe sample, into the receptacle vial. There may be multiple automatedassemblies in the same system.

The vial is lowered or otherwise placed back in its position on thereceptacle shuttle. A waste line is positioned in the fluid outlet path.Air is pumped through the FTA paper to clear the lines. The compressionram is retracted and the FTA card is removed from the flow throughdevice loaded back into magazine cassette. The FTA card may be imaged bythe imaging system before it is placedinto the cassette.

A clean FTA card (or clean portion of a previously sampled card) maythen be positioned within the isolation area and fluid flow path of theflow through device to allow cleaning of the system. The ram is extendedto compress FTA paper and solvent is pumped through the clean paperwhile the pressure is modulated to allow for controlled wicking/parallelsurface cleaning. Air is again pumped though the lines and paper toclear the lines. The compression ram is retracted and the clean papercard is removed from flow through device. This process may be repeatedas needed depending on the capacity of the system. For example, theprocess is repeated based on a certain number of samples (e.g. 12samples) on a vial strip, which is then returned to a strip shuttle, orbased a certain number of samples on a well plate (e.g. 96 samples),which is then returned to plate-stacker.

While only certain features of the invention have been illustrated anddescribed herein, many modifications and changes will occur to thoseskilled in the art. It is, therefore, to be understood that the appendedclaims are intended to cover all such modifications and changes as fallwithin the true spirit of the invention.

The invention claimed is:
 1. A system for processing samples fixed to aporous substrate comprising, a compressor defining one or more fluidisolation areas; a support, for the porous substrate, having an openingcorresponding to one or more of the fluid isolation areas of thecompressor and wherein the support extends away from the opening tosupport the porous substrate beyond the opening; an actuator that causesat least a portion of the compressor to press against the poroussubstrate from opposing surfaces to isolate only a portion of the poroussubstrate in one or more of the fluid isolation areas; a fluid inlethaving access to the fluid isolation area at least when the compressoris pressed against the porous substrate; and a fluid outlet to receivefluid, through the opening in the support corresponding to the fluidisolation area of the compressor, at least when the compressor ispressed against the porous substrate.
 2. The system of claim 1, whereinthe compressor comprises a top seal plug and a bottom seal plug, one orboth of which the actuator causes to press against the porous substrate.3. The system of claim 2, wherein the bottom seal plug has an opening influid communication with the opening in the support and an opening influid communication with the fluid outlet.
 4. The system of claim 2,wherein the top seal plug has an opening in fluid communication with thefluid inlet.
 5. The system of claim 1, further comprising a receptaclecomprising a plate having a plurality of wells.
 6. The system of claim1, further comprising a receptacle comprising one or more vials.
 7. Thesystem of claim 1, further comprising an analysis system.
 8. The systemof claim 7, wherein the analysis system comprises a liquidchromatography system.
 9. The system of claim 7, wherein the analysissystem comprises a mass spectrometry system.
 10. A system for processingsamples fixed to a porous substrate comprising, a plurality of sampleprocessing subsystems, each of which comprises, a compressor definingone or more fluid isolation areas; a support, for the porous substrate,having an opening corresponding to the fluid isolation area of thecompressor; an actuator that causes at least a portion of the compressorto press against the porous substrate to form a compressed fiber barrieragainst a second portion of the porous substrate that is outside thefluid isolation area; a fluid inlet having access to the fluid isolationarea at least when the compressor is pressed against the poroussubstrate; and a fluid outlet to receive fluid, through the opening inthe support corresponding to the fluid isolation area of the compressor,at least when the compressor is pressed against the porous substrate; amanifold in fluid communication with the fluid inlet of the sampleprocessing subsystems; a cassette for housing a plurality of poroussubstrates; a receptacle shuttle subsystem for transporting one or morefluid receptacles to the sample processing subsystems; an automatedassembly for transporting the porous substrates from the cassette to thesample processing subsystems; and a controller that coordinates thesample processing subsystems and the automated assembly.
 11. The systemof claim 10, further comprising an imaging subsystem for imaging thesamples on the porous substrates.
 12. The system of claim 10, whereinthe automated assembly comprises a robotic subassembly and a guide alongwhich the robotic subassembly moves between the cassette and the sampleprocessing systems.
 13. The system of claim 12, wherein the automatedassembly also transports the porous substrates to the imaging subsystem.14. A method for processing samples fixed to a porous substrate on asupport comprising, creating a compression seal around only a portion ofthe porous substrate, to form an isolation zone within which a portionof the sample is thereby isolated; applying a fluid to the sampleisolated in the isolation zone, where the fluid moves through the samplein a direction substantially perpendicular to the sample surface;collecting at least a portion of the fluid after it is applied to theisolation zone and analyzing one or both of the collected fluid and theportion of the sample in the isolation zone.
 15. The method of claim 14,further comprising clearing the fluid remaining in contact with theisolation zone.
 16. The method of claim 14, further comprising imagingthe sample on the porous substrate before creating the compression seal.17. The method of claim 16, further comprising imaging the sample afterthe fluid is collected.
 18. The method of claim 14, wherein theanalyzing step comprises quantifying an amount of one or more substancesin the collected fluid.
 19. The method of claim 14, wherein the samplecomprises blood and the analyzing step comprises identifying one or moredrug compound or drug metabolites in the sample.
 20. The method of claim14, wherein the sample comprises a biological material and the analyzingstep comprises determining one or more characteristics of the biologicalmaterial.
 21. The method of claim 14, wherein the creating thecompression seal comprises forming a plurality of isolation zones on theporous substrate.
 22. The method of claim 14, wherein a plurality offluids are applied to the sample.
 23. The method of claim 22, whereinthe plurality of fluids are applied simultaneously or serially.
 24. Themethod of claim 14, wherein a compressible membrane or filter is locatedbetween the porous substrate and the fluid outlet.
 25. The method ofclaim 24, wherein the compressible membrane or filter has an effectivepore size that is smaller than any fibers that may be released from thesample.
 26. A system for processing samples fixed to a porous substratecomprising, a compressor defining one or more fluid isolation areas; asupport, for the porous substrate, having an opening corresponding tothe fluid isolation area of the compressor; an actuator that causes atleast a portion of the compressor to press against the porous substratesuch that only a portion of the porous substrate is within the fluidisolation area and such that the compressor forms a compressed fiberbarrier to a second portion of the porous substrate that is outside thefluid isolation area; a fluid inlet having access to the fluid isolationarea at least when the compressor is pressed against the poroussubstrate; a fluid outlet to receive fluid, through the opening in thesupport corresponding to the fluid isolation area of the compressor, atleast when the compressor is pressed against the porous substrate; and aclearing component that clears one or more of the opening in thesupport, the fluid inlet or the fluid outlet.
 27. The system of claim26, wherein the clearing component clears by forcing a gas or a liquidthrough one or more of the opening in the support, the fluid inlet orthe fluid outlet.